CN114948852B - Microneedle system for brain disease diagnosis and treatment and preparation method thereof - Google Patents

Microneedle system for brain disease diagnosis and treatment and preparation method thereof Download PDF

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CN114948852B
CN114948852B CN202210589807.8A CN202210589807A CN114948852B CN 114948852 B CN114948852 B CN 114948852B CN 202210589807 A CN202210589807 A CN 202210589807A CN 114948852 B CN114948852 B CN 114948852B
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CN114948852A (en
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盛宗海
郑海荣
胡德红
高笃阳
刘新
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Shenzhen Institute of Advanced Technology of CAS
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    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
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    • A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
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    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
    • A61M2037/0061—Methods for using microneedles

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Abstract

The application relates to the technical field of medicine administration, and particularly discloses a microneedle system for brain disease diagnosis and treatment and a preparation method thereof. A microneedle system for brain disease diagnosis and treatment comprises 0.1-10 parts of active drug liposome; 1-10 parts of dispersing agent; wherein, the active drug liposome is prepared from the following raw materials: 0.1-10 parts of active medicine; 10-100 parts of phospholipid; cholesterol 10-100 parts. The preparation method comprises the following steps: is prepared through film hydration, freeze thawing and homogenization. The microneedle system for brain disease diagnosis and treatment has the advantages of changing the mode that the existing microneedle for brain disease treatment needs surgical implantation, adopting percutaneous administration, needing no surgery and having good treatment effect.

Description

Microneedle system for brain disease diagnosis and treatment and preparation method thereof
Technical Field
The application relates to the technical field of medical administration, in particular to a microneedle system for brain disease diagnosis and treatment and a preparation method thereof.
Background
Brain diseases are a type of heterogeneous neurological and psychiatric disorders occurring in the brain that cause serious personal pain and economic loss to the patient. Among various brain diseases, brain tumors represented by glioblastoma multiforme and brain neurodegenerative diseases represented by Alzheimer's disease and Parkinson's disease are particularly attracting attention. The number of patients is high in the world, the disease is serious, and the existing medicine has unsatisfactory diagnosis and treatment effects. The natural defense system of the brain, the blood brain barrier (Blood Brain Barrier, BBB), protects the brain from harmful substances, and simultaneously limits the entry of most imaging probes and drug molecules, thereby severely affecting the diagnosis and treatment of glioma.
The lymphatic system is another set of fluid reticulation circulatory system independent of the blood circulatory system, which extends over most tissues of the human body and assists in the removal of metabolic waste products from the interstitium to maintain humoral homeostasis and to develop immune responses and immune monitoring. Although meningeal lymphatic vessels have a certain specificity in development time and morphology, they are markers of high expression of mature lymphatic endothelial cells, similar to peripheral lymphatic vessels. The function of the cerebral lymphatic system is not limited to the removal of metabolites, but the effect on the cerebrospinal fluid circulation is also not negligible. The university of 1 month 15 yersil medical school Akiko Iwasaki professor team in Nature journal published articles reveal that vascular endothelial growth factor C (VEGF-C) promotes the drainage of CD8T cells into the brain in deep cervical lymph nodes and promotes the activation of CD8T cells, migration to tumor sites, rapid clearance of glioblastoma, and long lasting anti-tumor immune memory response. However, after ligation of the deep cervical lymph nodes, this anti-tumor effect of VEGF-C was lost, indicating that clearance of glioblastoma by VEGF-C requires lymphatic drainage to the deep cervical lymph nodes. Therefore, the development of a safe and efficient novel brain drug delivery strategy for the deep-neck lymph nodes becomes an important research target in the field of central nervous systems.
The micro-needle can overcome the stratum corneum obstacle in a minimally invasive and painless way, can effectively promote the percutaneous permeation of drug molecules, and has remarkable effect in the aspect of percutaneous delivery of macromolecular drugs. In the beginning of the 21 st century, there were increasing experiments demonstrating that microneedles can greatly enhance transdermal delivery of insulin. With the increasing abundance of materials, more and more materials are being developed for the preparation of insulin microneedles. Among them, soluble insulin microneedles have received a great deal of attention because they can achieve an administration effect similar to injection. The team of ceramic tiger researchers at Shanghai microsystems of Chinese academy of sciences cooperates with the professor team of neurosurgery Mao Ying of the China mountain hospital affiliated to the compound denier university to develop heterogeneous and degradable microneedle patches based on fibroin aiming at the treatment of brain glioma. The microneedle patch can carry three medicines simultaneously, the release sequence and the release period of the medicines can be matched with the difference requirement of clinical medication standards, and the microneedle patch has the key functions of rapid hemostasis in operation, tumor cell inhibition by long-term chemotherapy after operation, targeted angiogenesis inhibition by timing start as required and the like. In the operation process of cutting tumor, the patch is implanted in situ into tumor cavity, and can be completely degraded and disappeared after releasing the medicine, without taking out by secondary operation, and the degradation product can not cause immune inflammatory reaction. The intracranial local administration mode not only solves the problem of the obstruction of the blood brain barrier to the drug molecules, but also reduces the toxic and side effects of the conventional systemic large-dose administration. In animal experiments, compared with blank groups and injection groups, the treatment groups adopting the fibroin drug-loaded microneedle patch effectively inhibit tumor volume and remarkably prolong survival time of mice. The university of john hopkins Li Xingde teaches a subminiature (580 μm outer diameter) therapeutic deep brain microneedle combined with 800nm optical coherence tomography with laser ablation. The effectiveness of this method was demonstrated by in vivo ultra-high resolution (axial 1.7 μm, lateral 5.7 μm), high-speed (20 frames per second) volumetric imaging of the mouse brain microstructure and determination of the light attenuation coefficient. The transformation potential was further demonstrated by in vivo tumor visualization (imaging depth of 1.23 mm) and efficient tissue ablation (350 mw power 1448nm continuous wave laser) in the deep brain of mice.
However, these above methods all require the microneedle to be surgically implanted into brain tissue, and it is well known that there is a certain risk in surgery regardless of size, and in other cases, there is an increased risk for brain surgery. The inventors consider that the related art has a single administration mode and a high risk for a brain disease treatment site.
Disclosure of Invention
In order to overcome the defects of single administration mode and high operation risk of the micro-needle for diagnosing and treating the brain diseases in the related art, the application provides a micro-needle system for diagnosing and treating the brain diseases and a preparation method thereof.
In a first aspect, the present application provides a microneedle system for brain disease diagnosis and treatment, which adopts the following technical scheme:
a microneedle system for brain disease diagnosis and treatment comprises the following raw materials in parts by weight:
0.1-10 parts of active drug liposome;
1-10 parts of dispersing agent;
Wherein, the active drug liposome is prepared from the following raw materials:
0.1-10 parts of active medicine;
10-100 parts of phospholipid;
Cholesterol 10-100 parts.
By adopting the technical scheme, the microneedle loaded with the active drug liposome can successfully deliver the active drug liposome to the brain through the deep cervical lymph node, thereby exerting the therapeutic effect. Can avoid the interference of gastrointestinal environment to drug effect and liver first pass effect, maintain constant optimal blood concentration or physiological effect, prolong effective action time, reduce administration times, and has independent administration and good compliance.
Optionally, the microneedle system for brain disease diagnosis and treatment further comprises NK cell membrane proteins.
By adopting the technical scheme, NK cells are Natural killer cells (Natural KILLER CELL, NK), are important immune cells of organisms, are not only related to anti-tumor, anti-virus infection and immune regulation, but also are involved in hypersensitivity and autoimmune diseases in certain cases; the membrane protein is adopted for bionic, so that the rate of the active drug liposome entering brain cells can be effectively improved, and the effect is rapidly exerted.
Optionally, the mass ratio of the active drug liposome to the NK cell membrane protein is 300:1.
Optionally, the active drug liposome and NK cell membrane protein are prepared into bionic active drug liposome by a homogenization method, and the specific operation of the homogenization method is as follows:
1) Homogenizing under 20psi pressure for 3-10 times;
2) Regulating air pressure to 40psi, and homogenizing for 3-10 times to obtain bionic active pharmaceutical liposome.
By adopting the technical scheme, the active drug liposome and NK cell membrane protein are enabled to be suitable in particle size, uniform in particle size and smooth in surface without crystallization through a high-pressure homogenization method.
Optionally, the active drug comprises a water-soluble active drug and a fat-soluble active drug; when the active drug is a liposoluble active drug, the preparation of the liposoluble active drug liposome comprises the following steps:
s1, preparation of liposoluble active medicine carrier,
Preparation of a liposoluble active drug carrier: dissolving 10-100 parts of phospholipid and 10-100 parts of cholesterol in chloroform to obtain a mixed solution I for standby;
the fat-soluble drug pretreatment method comprises the following steps: taking 0.1-10 parts of fat-soluble active medicine, and dissolving the fat-soluble active medicine in absolute methanol or diethyl ether to obtain a mixed solution II for standby;
S2, steaming the mixed solution in a rotary way,
Mixing the mixed solution I and the mixed solution II, and then evaporating to dryness to obtain a mixture;
s3, preparing active drug liposome,
1) Flushing the mixture by using PBS buffer solution, then performing freeze thawing at the temperature of liquid nitrogen-65 ℃, and circulating for 4-8 times to obtain active drug liposome solution;
2) Dialyzing or squeezing and filtering, dialyzing to obtain active drug liposome, and storing at 4deg.C in dark place;
when the active drug is a water-soluble active drug, the preparation of the water-soluble active drug liposome comprises the following steps:
S1, preparation of a water-soluble active drug carrier: dissolving 10-100 parts of phospholipid and 10-100 parts of cholesterol in chloroform to obtain a mixed solution I for standby;
s2, rotary steaming the mixed solution: evaporating the mixed solution I by rotary evaporation to dryness to obtain a mixture;
s3, preparing active drug liposome,
1) Washing the mixture by using PBS buffer solution, adding 0.1-10 parts of water-soluble active medicine to fully dissolve the mixture, then performing freeze thawing at the temperature of liquid nitrogen-65 ℃, and circulating for 4-8 times to obtain active medicine liposome solution;
2) Dialyzing or squeezing and filtering, dialyzing to obtain active pharmaceutical liposome, and storing at 4deg.C in dark place.
By adopting the technical scheme, firstly, chloroform is used for dissolving phospholipid and cholesterol, absolute methanol and diethyl ether are used for dissolving active drugs, then the active drugs are mixed, so that the active drugs can be fully mixed with the phospholipid and the cholesterol, the active drugs can be uniformly distributed in liposome, secondly, PBS is used for flushing the mixture to be flushed and shed from the container wall, and the last step of S3 is used for dialysis to remove free active drugs; wherein the liposoluble drugs such as curcumin, nimodipine, flunarizine, ligustrazine, papaverine, vinpocetine, etc. are dissolved by anhydrous methanol, and then dispersed in liposome, such as water-soluble drugs such as betahistine, sodium valproate, phenytoin sodium, etc., and then dissolved directly by PBS buffer.
Preferably, the active drug includes a water-soluble active drug and a fat-soluble active drug; when the active drug is a liposoluble active drug, the preparation of the liposoluble active drug liposome comprises the following steps:
s1, preparation of liposoluble active medicine carrier,
Preparation of a liposoluble active drug carrier: dissolving 50 parts of phospholipid and 25 parts of cholesterol in chloroform to obtain a mixed solution I for standby;
the fat-soluble drug pretreatment method comprises the following steps: taking 0.25 part of fat-soluble active medicine, and dissolving the fat-soluble active medicine in absolute methanol or diethyl ether to obtain a mixed solution II for standby;
S2, steaming the mixed solution in a rotary way,
Mixing the mixed solution I and the mixed solution II, and then evaporating to dryness to obtain a mixture;
s3, preparing active drug liposome,
1) Washing the mixture by using PBS buffer solution, then performing freeze thawing at the temperature of liquid nitrogen-65 ℃, and circulating for 6 times to obtain active drug liposome solution;
2) Dialyzing or squeezing and filtering, dialyzing to obtain active drug liposome, and storing at 4deg.C in dark place;
When the active drug is a water-soluble active drug, the preparation of the water-soluble active drug liposome comprises the following steps: s1, preparation of a water-soluble active drug carrier: dissolving 50 parts of phospholipid and 25 parts of cholesterol in chloroform to obtain a mixed solution I for standby;
s2, rotary steaming the mixed solution: evaporating the mixed solution I by rotary evaporation to dryness to obtain a mixture;
s3, preparing active drug liposome,
1) Washing the mixture by using PBS buffer solution, adding 0.25 part of water-soluble active medicine to fully dissolve the water-soluble active medicine, then performing freeze thawing at the temperature of liquid nitrogen-65 ℃, and circulating for 5 times to obtain active medicine liposome solution;
2) Dialyzing or squeezing and filtering, dialyzing to obtain active pharmaceutical liposome, and storing at 4deg.C in dark place.
Optionally, the phospholipid is soybean lecithin.
Optionally, the extruding operation in step 2) in S3 is specifically: repeatedly extruding for 20 times by adopting a liposome extruder provided with filter membranes with different pore diameters, and sequentially reducing the pore diameters of the filter membranes.
Alternatively, the filter membrane is a polycarbonate membrane, and the pore size of the polycarbonate membrane is 200nm,100nm and 50nm.
Specifically, the extrusion operation in step 2) in S3 is to sequentially perform extrusion filtration by using a liposome extruder equipped with polycarbonate membranes with pore diameters of 200nm,100nm and 50nm, and the extrusion filtration times are 20 times respectively.
By adopting the technical scheme, particles in the liposome are crushed through repeated extrusion and filtration, and sequentially pass through 200nm, 100nm and 50nm filter membranes, so that the particle size of the liposome is uniform, and finally the active drug liposome with the particle size of 50nm is obtained.
Optionally, the dispersing agent is one or more of sodium hyaluronate, hyaluronic acid, dextran, or polyethylene glycol (PEG).
Preferably, the dispersing agent is sodium hyaluronate and dextran.
Optionally, the active drug is one of curcumin, nimodipine, flunarizine, betahistine, ligustrazine, butylphthalide, papaverine, ginkgo leaf extract, vinpocetine, quetiapine fumarate, olanzapine, citalopram, alprazolam, oxazepam (nordiazepam), lorazepam (roller), triazolam (halreshen), methoabane, tiazem Shu Da, feflozin, rest, selegiline, rasagiline, tolcapone, ropinirole, ambrisen, ambroxol, copad, amantadine, carbamazepine, sodium valproate, sodium phenytoin, gabapentin, lamotrigine, oxzepine, phenobarbital, topiramate, vigabatrin, levetiracetam, clonazepam, idamole maleate, ethylsuone, epileptine, zolamide, pregabane, and tegafur.
In a second aspect, the present application provides a method for preparing a microneedle system for diagnosing and treating brain diseases, which adopts the following technical scheme: a method of preparing a microneedle system for brain disease diagnosis and treatment, comprising the steps of:
1) Lyophilizing the active drug liposome or bionic active drug liposome to obtain white powder;
2) Dispersing 0.1-10 parts of active drug liposome or bionic active drug liposome and 1-10 parts of dispersing agent in water in 1), and stirring until the active drug liposome or bionic active drug liposome and the dispersing agent are fully dissolved to obtain matrix liquid;
3) Injecting the matrix liquid obtained in the step 2) into a microneedle mould, and performing centrifugal inversion to uniformly distribute the matrix liquid in the mould and fill the microneedle tip part in the mould;
4) Dispersing 1-10 parts of dispersing agent in water to obtain dispersion liquid; then adding the dispersion liquid into the microneedle tips in the step 3), centrifuging and drying to obtain active drug liposome microneedles or bionic active drug liposome microneedles;
5) And (3) sticking a pressure-sensitive adhesive lining on the back surface of the microneedle substrate, and demolding to obtain the active drug liposome microneedle patch or bionic active drug liposome microneedle patch.
Preferably, a method for preparing a microneedle system for brain disease diagnosis and treatment, comprising the following preparation steps:
1) Lyophilizing the active drug liposome or bionic active drug liposome to obtain white powder;
2) Dispersing 2 parts of active drug liposome or bionic active drug liposome and 4 parts of dispersing agent in water in 1), and stirring until the active drug liposome or bionic active drug liposome and the dispersing agent are fully dissolved to obtain matrix liquid;
3) Injecting the matrix liquid obtained in the step 2) into a microneedle mould, and performing centrifugal inversion to uniformly distribute the matrix liquid in the mould and fill the microneedle tip part in the mould;
4) Then 1 part of glucan and 2 parts of sodium hyaluronate are taken and dispersed in water to obtain dispersion liquid; then adding the dispersion liquid into the microneedle tips in the step 3), centrifuging and drying to obtain active drug liposome microneedles or bionic active drug liposome microneedles;
5) And (3) sticking a pressure-sensitive adhesive lining on the back surface of the microneedle substrate, and demolding to obtain the active drug liposome microneedle patch or bionic active drug liposome microneedle patch.
Optionally, the drying temperature in the step 4) is 4 ℃, and the drying time is 1-100h; preferably, the drying time is 24 hours.
In summary, the application has the following beneficial effects: the microneedle system for brain disease diagnosis and treatment provided by the application does not need an operation, and is an implantable slow-release microneedle which is simple in process, high in safety and free from long-term application. Transdermal administration (applied to the head or neck) is adopted, so that the medicine passes through the deep lymph node and meningeal lymphatic vessel of the neck, bypasses the blood brain barrier and reaches brain tissue, and the therapeutic effect is exerted.
Drawings
FIG. 1 is a TEM image of curcumin liposomes of a microneedle system for brain disease diagnosis and treatment disclosed in the present application;
FIG. 2 is a graph of particle size of curcumin liposome of a microneedle system for brain disease diagnosis and treatment disclosed in the present application;
FIG. 3 is a curcumin profile of a mouse brain slice in example 1 of the present application;
FIG. 4 is a table showing comparison of curcumin efficiencies of application examples 1 to 3 of the present application with respect to different time points into the brain.
Detailed Description
The application aims to overcome the defects in the prior art and provide an in-vitro brain or neck microneedle patch for treating brain diseases. The method loads the active medicine for treating brain diseases into the micro-needle, and explores the percutaneous administration effect (attached to the head or neck) of the micro-needle loaded with the active medicine. In other embodiments, the active drug may be nimodipine, flunarizine, betahistine, ligustrazine, butylphthalide, papaverine, ginkgo biloba extract, vinpocetine, quetiapine fumarate, olanzapine, citalopram, alprazolam, oxazepam (nordiazepam), lalazepam (roller), triazolam (halibut), metaba, tiazem Shu Da, senformol, rest, tranexamine, selegiline, rasagiline, tolcapone, ropinirole, ambam, ambroxol, collotype, amantadine, carbamazepine, sodium valproate, phenytoin sodium, gabapentin, lamotrigine, oxcarbazepine, phenobarbital, topiramate, vigabatrin, clonazepam, diazepam, macarbazem, zamide, bazepam, zamide, pam, and the like.
The results show that the microneedle loaded with the curcumin liposome can successfully deliver the curcumin liposome to the brain through the deep cervical lymph node, and exert the therapeutic effect. The invention has important significance for promoting the application and transformation of the microneedles for clinically treating the brain diseases, and provides a new method and a new technology for treating the brain diseases.
The application is described in further detail below with reference to fig. 1 to 4 and examples. The specific description is as follows: the following examples, in which the specific conditions are not specified, are conducted under conventional conditions or conditions recommended by the manufacturer, and the raw materials used in the following examples are commercially available from ordinary sources except for the specific descriptions.
Preparation example of curcumin liposome
Preparation example 1
S1, curcumin dissolution and curcumin carrier preparation,
Preparation of curcumin carrier: dissolving 50mg of phospholipid and 25mg of cholesterol in 2ml of chloroform to obtain a mixed solution I for standby;
Curcumin pretreatment: dissolving 0.25mg of curcumin in 2ml of absolute methanol to obtain a mixed solution II for standby;
s2, steaming the mixed solution in a rotary way, mixing the mixed solution I and the mixed solution II in a round-bottom flask, and then putting the round-bottom flask in a rotary steaming instrument (model is IKA-RV 10) for rotary steaming, wherein the rotary speed of the rotary steaming instrument is 60rpm, the temperature is 27 ℃, and the wall of the round-bottom flask is steamed to form a film mixture;
S3, preparing curcumin liposome,
1) Washing the film mixture on the wall of the flask with 5ml of PBS to completely fall off, and then performing freeze thawing at the temperature of liquid nitrogen-65 ℃ for 5 times to obtain liposome solution;
2) Repeatedly squeezing for 20 times by using a liposome squeezer (model Avanti liposome squeezer 610023) with a polycarbonate membrane, and sequentially filtering; wherein the polycarbonate film has a pore size of 200nm, 100nm and 50nm; the pore size of the filter membrane used in extrusion filtration is sequentially reduced so as to obtain liposome with uniform particle size
3) Finally, the curcumin liposome is prepared by dialysis and is preserved in a dark place at the temperature of 4 ℃.
Preparation examples 2 to 3 differ from preparation example 1 in the amounts of the raw materials used, in particular in the following table.
Preparation example of bionic curcumin liposome
Preparation example 4
30Mg of the curcumin liposome prepared in preparation example 1 was placed in a syringe, and then 0.1mg of NK cell membrane protein was added to the syringe.
Opening a high-pressure homogenizer air pump, setting the pressure to 20psi, and opening a homogenizing valve filling pressure; loading the syringe with the sample into a homogenizer, repeating the homogenization operation for 5 times, setting the pressure to 40psi, continuing the homogenization operation, and repeating the homogenization operation for 5 times to obtain the bionic curcumin liposome.
Examples
Example 1
1) Lyophilizing curcumin liposome prepared in preparation example 1 to obtain white powder;
2) Dispersing 2g of curcumin liposome or bionic curcumin liposome in 1) and 4g of sodium hyaluronate in 4ml of water, and stirring until the curcumin liposome or bionic curcumin liposome and the sodium hyaluronate are fully dissolved to obtain matrix liquid;
3) Injecting the matrix liquid obtained in the step 2) into a microneedle mould, and performing centrifugal inversion for 6 times to uniformly distribute the matrix liquid in the mould and fill the microneedle tip part in the mould;
4) Then 1g of glucan and 2g of sodium hyaluronate are taken and dispersed in 7ml of water to obtain dispersion liquid; then adding the dispersion liquid into the tip of the microneedle in the step 3), centrifuging and drying for 24 hours to obtain the curcumin liposome microneedle;
5) And (5) sticking a pressure-sensitive adhesive lining on the back surface of the microneedle substrate, and demolding to obtain the curcumin liposome microneedle patch.
Examples 2 to 3
Examples 2-3 differ from example 1 in the amounts of raw materials used, as shown in the following table.
Example 4
This example differs from example 1 in that the biomimetic curcumin liposome prepared in preparation example 4 was used. And (3) scanning the obtained bionic curcumin liposome by an electron microscope to obtain a TEM image of the bionic curcumin liposome shown in the figure 1, wherein the TEM image shows that the particle size of the bionic curcumin liposome is about 50nm, and the dispersity is relatively good. In addition, the particle size distribution of the bionic curcumin liposome is measured by a particle size analyzer, and as shown in fig. 2, the hydration particle size of the bionic curcumin liposome is about 50nm, and the particles are relatively uniform.
Application example
Application example 1: firstly, taking an experimental mouse, and removing hairs on the neck and the head of the mouse; the curcumin liposome microneedle patch prepared in example 1 was then applied to the neck of mice (curcumin liposome group); finally, the distribution of curcumin in the brain of mice was followed using the in vivo small animal imaging system (IVIS), as shown in fig. 3 and 4.
Application example 2: the difference from application example 1 is that the bionic curcumin liposome microneedle patch prepared in example 4 was attached to the neck of a mouse (bionic curcumin liposome array), and the distribution of curcumin in the brain of the mouse was tracked using a small animal In Vivo Imaging System (IVIS), as shown in fig. 4.
Application example 3: the curcumin liposome obtained in preparation example 1 was injected into the tail vein (tail vein group) of a mouse, and then the distribution of curcumin in the brain of the mouse was tracked using a small animal In Vivo Imaging System (IVIS), as shown in fig. 4.
In other applications, the curcumin liposome or the bionic curcumin liposome prepared by the preparation method can be made into gel to be attached to the head or the neck, or introduced into the head or the neck by adopting methods such as ultrasonic and subcutaneous injection.
In combination with the embodiment and the application example, fig. 1 to fig. 4 can show that the microneedle patch prepared by the preparation method of the microneedle system provided by the application shows that the microneedle loaded with the curcumin liposome or the bionic curcumin liposome can successfully deliver the curcumin liposome or the bionic curcumin liposome to the brain through the deep cervical lymph node, thereby exerting the therapeutic effect. The application has important significance for promoting the application and transformation of the microneedle of clinical curcumin liposome and provides a new method and a new technology for treating brain diseases. Compared with the traditional brain disease treatment method, the method does not need operation, reduces the treatment risk, has simple process and high safety, and does not need long-term application. The medicine can avoid the interference of gastrointestinal environment to medicine effect and the first pass effect of liver, maintain constant optimal blood concentration or physiological effect, prolong effective action time, reduce medicine taking times, and ensure that patients can take medicine independently.
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.

Claims (4)

1. The microneedle patch for brain disease diagnosis and treatment is characterized by comprising the following raw materials in parts by weight:
0.1-10 parts of curcumin liposome;
1-20 parts of dispersing agent;
the curcumin liposome is prepared from the following raw materials in parts by weight:
curcumin 0.1-10 parts
10-100 Parts of phospholipid;
10-100 parts of cholesterol;
The dispersing agent is one or a combination of more of sodium hyaluronate, hyaluronic acid, dextran or PEG;
The preparation of the curcumin liposome comprises the following steps:
S1, curcumin pretreatment and curcumin carrier preparation,
Preparation of curcumin carrier: dissolving 10-100 parts of phospholipid and 10-100 parts of cholesterol in chloroform to obtain a mixed solution I for standby;
The curcumin pretreatment method comprises the following steps: dissolving 0.1-10 parts of curcumin in absolute methanol or diethyl ether to obtain a mixed solution II for standby;
S2, steaming the mixed solution in a rotary way,
Mixing the mixed solution I and the mixed solution II, and then evaporating to dryness to obtain a mixture;
S3, preparing curcumin liposome,
1) Washing the mixture by using PBS buffer solution, then performing freeze thawing at the temperature of liquid nitrogen-65 ℃, and circulating for 4-8 times to obtain curcumin liposome solution;
2) Dialyzing or squeezing and filtering, dialyzing to obtain curcumin liposome, and storing at 4deg.C in dark place;
the preparation of the microneedle patch for brain disease diagnosis and treatment comprises the following preparation steps:
1) Lyophilizing the curcumin liposome to obtain white powder;
2) Dispersing 0.1-10 parts of curcumin liposome and 1-10 parts of dispersing agent in water in 1), and stirring until the curcumin liposome and the dispersing agent are fully dissolved to obtain matrix liquid;
3) Injecting the matrix liquid obtained in the step 2) into a microneedle mould, and performing centrifugal inversion to uniformly distribute the matrix liquid in the mould and fill the microneedle tip part in the mould;
4) Dispersing 1-10 parts of dispersing agent in water to obtain dispersion liquid; then adding the dispersion liquid into the tip of the microneedle in the step 3), centrifuging and drying to obtain the curcumin liposome microneedle;
5) And (5) sticking a pressure-sensitive adhesive lining on the back surface of the microneedle substrate, and demolding to obtain the curcumin liposome microneedle patch.
2. The microneedle patch for brain disease diagnosis and treatment is characterized by comprising the following raw materials in parts by weight:
0.1-10 parts of curcumin liposome;
1-20 parts of dispersing agent;
NK cell membrane proteins;
wherein, the mass ratio of the curcumin liposome to the NK cell membrane protein is 300:1;
the curcumin liposome is prepared from the following raw materials in parts by weight:
curcumin 0.1-10 parts
10-100 Parts of phospholipid;
10-100 parts of cholesterol;
The dispersing agent is one or a combination of more of sodium hyaluronate, hyaluronic acid, dextran or PEG;
The preparation of the curcumin liposome comprises the following steps:
S1, curcumin pretreatment and curcumin carrier preparation,
Preparation of curcumin carrier: dissolving 10-100 parts of phospholipid and 10-100 parts of cholesterol in chloroform to obtain a mixed solution I for standby;
The curcumin pretreatment method comprises the following steps: dissolving 0.1-10 parts of curcumin in absolute methanol or diethyl ether to obtain a mixed solution II for standby;
S2, steaming the mixed solution in a rotary way,
Mixing the mixed solution I and the mixed solution II, and then evaporating to dryness to obtain a mixture;
S3, preparing curcumin liposome,
1) Washing the mixture by using PBS buffer solution, then performing freeze thawing at the temperature of liquid nitrogen-65 ℃, and circulating for 4-8 times to obtain curcumin liposome solution;
2) Dialyzing or squeezing and filtering, dialyzing to obtain curcumin liposome, and storing at 4deg.C in dark place;
the curcumin liposome and NK cell membrane protein prepared by the method are prepared into bionic curcumin liposome by a homogenization method, and the homogenization method comprises the following specific operations: 1) Homogenizing under 20psi pressure for 3-10 times; 2) Regulating air pressure to 40psi, and continuing homogenizing for 3-10 times to obtain bionic curcumin liposome;
the preparation of the microneedle patch for brain disease diagnosis and treatment comprises the following preparation steps:
1) Lyophilizing the bionic curcumin liposome to obtain white powder;
2) Dispersing 0.1-10 parts of bionic curcumin liposome and 1-10 parts of dispersing agent in water in 1), and stirring until the bionic curcumin liposome and the dispersing agent are fully dissolved to obtain matrix liquid;
3) Injecting the matrix liquid obtained in the step 2) into a microneedle mould, and performing centrifugal inversion to uniformly distribute the matrix liquid in the mould and fill the microneedle tip part in the mould;
4) Dispersing 1-10 parts of dispersing agent in water to obtain dispersion liquid; then adding the dispersion liquid into the tip of the microneedle in the step 3), centrifuging and drying to obtain the bionic curcumin liposome microneedle;
5) And (5) sticking a pressure-sensitive adhesive lining on the back surface of the microneedle substrate, and demolding to obtain the bionic curcumin liposome microneedle patch.
3. A microneedle patch for use in the diagnosis and treatment of brain diseases according to claim 1or 2, wherein S3 further comprises squeeze filtration, the squeeze filtration operation being specifically: repeatedly extruding for 20 times by adopting a liposome extruder provided with filter membranes with different pore diameters in sequence, wherein the pore diameters of the filter membranes are reduced in sequence.
4. A microneedle patch for the diagnosis and treatment of brain diseases according to claim 3, wherein the filter membrane is a polycarbonate membrane having pore diameters of 200nm,100nm and 50nm.
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